The Trials and Tribulations of Vitamin C Stephen Lawson1 Introduction Ascorbic acid is a small water-soluble molecule that functions as a vitamin in a few species, including guinea pigs, humans, and other primates. Although citrus fruits were recognized as a dietary imperative to prevent scurvy during sea voyages of the 18th century, the anti-scorbutic factor, vitamin C, was not isolated until 1928, when Albert Szent-Györgyi extracted vitamin C from animal adrenal glands. The chemical structure of vitamin C, which has the formula C6H8O6, was determined by Haworth and Hirst in 1933.1 Vitamin C functions as an electron donor for a number of enzymes, including those involved in the synthesis of collagen, carnitine, hormones, and neurotransmitters. Vitamin C also enhances the detoxification of xenobiotics and carcinogens by cytochrome P450 enzymes, may be required for immunocompetence, and is involved in the conversion of cholesterol to bile acids. Vitamin C is the most effective aqueous antioxidant in plasma,2 scavenging reactive oxygen species and reactive nitrogen species.3,4 The Media and the Safety of Vitamin C In the last few years, a number of reports have appeared in the scientific literature that raised concerns about the safety of vitamin C. Despite the many published safety reviews that have found few, if any, problems associated with the intake of supplemental vitamin C. 5-8 the media have unduly emphasized this spate of negative reports without examining them in the context of the accumulated scientific literature. The public has been left with uncertainty about the safety of the routine use of large doses of vitamin C for prophylactic or therapeutic purposes. Recently, the news media mischarac1. Linus Pauling Institute, Oregon State University 571 Weniger Hall, Corvallis, OR 97333

terized the information on vitamin C in the report by the British Expert Group on Vitamins and Minerals (EVM) on the safe upper levels for vitamins and minerals.9 For instance, on May 7, 2003, the BBC stated that “people who take large doses of certain vitamins and minerals risk permanently damaging their health, a government watchdog has warned.” The article continued, “Experts from the UK’s Food Standards Agency say high levels of minerals like beta-carotene [sic] and zinc over a long period may have irreversible harmful effects…In addition, they reiterated warnings that high doses of vitamin C, calcium and iron can harm health but said longterm damage can be avoided if people stop taking them.” The following day, the BBC reported that “The FSA is also warning that too much vitamin C, calcium and iron in supplement form may be damaging.” An examination of the EVM report reveals that no such strong warnings on vitamin C were issued. The EVM report stated that “The available data suggest that vitamin C is not associated with significant adverse effects and there are no obvious specific key toxic endpoints for vitamin C dose given orally to healthy subjects.” The report continued, “There are insufficient data to set a Safe Upper Level for vitamin C. The vitamin may be of low toxicity, though adverse effects, in particular on the gastrointestinal system, may occur in subjects consuming quantities of vitamin C greater than 1000 mg/day… It should be noted that higher levels of vitamin C may be without adverse effects in many individuals.” This viewpoint is concordant with that expressed by the U.S. Institute of Medicine in 2000, which could not identify any serious side effects of vitamin C and set the tolerable upper intake level (UL) at 2,000 mg/day based solely on the possible laxative effect of vitamin C at larger doses, a side effect easily rectified by

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decreasing the dose.7 The EVM report concluded with the speculation that subjects with hemochromatosis, thalassemia, or disordered gastrointestinal function may be harmed by large doses of vitamin C. Johnston evaluated biomarkers for setting a UL for vitamin C in 1999.10 After carefully reviewing the potential adverse effects of large doses of vitamin C on systemic conditioning, kidney stone formation, pro-oxidant activity, iron overload, hemolysis in subjects with glucose-6-phosphate dehydrogenase deficiency, and destruction of vitamin B12, Johnston concluded that “the available data indicate that oral intakes of very high amounts of vitamin C (2-4 g/day) are well tolerated biologically. Presently, strong scientific evidence to define and defend a UL for vitamin C is not available.” A recurrent concern about supplemental vitamin C is its putative role in kidney stone formation. This has been very effectively addressed by two large-scale prospective studies that investigated the association between vitamin C intake and kidney stone formation.11,12 The first cohort of 45,251 men aged 40-75 years with no history of kidney stones was followed for 6 years. There was no increased risk for stone formation in men with the highest daily intake of vitamin C (≥1,500 mg) compared to those with the lowest daily intake (490 mg/day) were strongly associated with protection against cataract. In one study of 50,828 women, long-term supplementation (≥10 years) with vitamin C was also associated with a 45% reduced risk of cataracts, whereas shorter duration of vitamin C use was not. More recently, the ARED study of 4,757 subjects reported no difference in agerelated cataract incidence or progression in those taking a daily antioxidant cocktail containing 500 mg of vitamin C, 400 IU of vitamin E, 15 mg of beta-carotene, and with or without 80 mg of zinc oxide and 2 mg of cupric oxide for an average of 6.3 years compared to the placebo group.49 However another arm of the ARED study with 3,640 subjects found that the supplemented group had a significantly reduced risk for the development of advanced age-related macular degeneration.50 Finally, Carr and Frei reviewed the effect of vitamin C given orally (500 or 2,000 mg) or by intraarterial infusion (10-25 mg/min, 1,000 mg or 3,000 mg) on vasodilation. All twelve of the clinical studies reported significantly improved vasodilation following vitamin C administration, probably due 178

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to the stabilization of tetrahydrobiopterin, which increases the synthesis of nitric oxide in endothelial cells. Improved vasodilation following vitamin C supplementation may also be useful in preventing some of the complications of diabetes. In a number of intervention studies, supplemental vitamin C in doses of 500 mg or 1,000 mg has also been found to significantly lower systolic and/or diastolic blood pressure in borderline hypertensive subjects,51-54 although one study of patients with Type II diabetes did not find any effect of 1,500 mg/day of vitamin C on blood pressure or endothelial function.55 One non-randomized depletion/repletion study of 68 healthy subjects, including 4 hypertensives, found that plasma vitamin C was inversely associated with diastolic blood pressure.56 Subjects in this study were given a relatively small daily dose of vitamin C (117 mg) for one month. Pauling reported three case studies in which the provision of 3-6 grams/day each of L-lysine and vitamin C rapidly ameliorated severe exercise-induced angina pectoris in patients with severe coronary artery disease.57,58,59 While L-lysine may have exerted effects unrelated to vasodilation, such as binding to Lp(a) as hypothesized by Pauling, some of the observed benefit may have been due to the vasodilatory effects of vitamin C. There has been speculation about the utility of antioxidant vitamins used adjunctively in surgery. In a randomized, prospective study of 595 subjects, Nathens et al recently reported that the risk of pulmonary morbidity, organ failure and length of ICU stay were substantially reduced in surgical trauma patients who received 1,000 IU of vitamin E and 1,000 mg of intravenous vitamin C per day for the duration of admission to the ICU or 28 days, compared to similar patients not receiving the supplements.60 Cancer In 1999, a paper on the accumulation of vitamin C in cancer cells raised speculation about the possible harmful effects of

supplemental vitamin C in cancer patients. Agus et al. reported that dehydroascorbic acid (DHA), possibly formed pericellularly by the oxidative burst of neighboring activated neutrophils,61 is taken up by human xenograft tumor cells in athymic mice by the facilitative glucose transporters (GLUTs) and is then reduced to ascorbic acid, which is trapped intracellularly and accumulates.62 The investigators speculated that the high intracellular vitamin C concentration might be exploited by cancer cells. The import of these observations is questionable, since DHA is virtually undetectable in the plasma of healthy humans and may be out-competed for cellular transport by glucose.63 Other research has shown that vitamin C, but not DHA, enters cells through two sodiumdependent vitamin C transporters with different tissue distribution, SVCT1 (present in epithelial cells of the intestine, kidney, and liver), whose efficiency appears to be age-related64 and SVCT2 (found in a wide range of tissues and organs, including the eye and brain).65,66 In 1997, Agus et al. showed that DHA crosses the blood-brain barrier in rats and mice, a process mainly mediated by facilitative glucose transporters.67 In these experiments, 14C-labelled DHA and ascorbic acid were injected into the tail vein of animals. DHA was found in the brain after a short period, and ascorbic acid appeared after 30 minutes. Again, the physiological relevance of these observations is questionable, since DHA is not normally detectable in plasma. The relative contribution of the different transport mechanisms for vitamin C in different cell types remains unresolved. Many in vitro and in vivo studies have demonstrated an anticancer effect of vitamin C and its metabolites, suggesting that the cytotoxic effect of vitamin C predominates. Of course, vitamin C in vitro is readily oxidized by trace amounts of free transition metal ions in the culture me-

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dium, so recognition of the importance of the use of metal chelators is paramount in interpreting cell culture experiments using vitamin C. Tsao et al. investigated the cytotoxic effects of ascorbate derivatives on mouse leukemia cells and found that the cytotoxic moiety resides in the enediol lactone ring of the various derivatives.68 Similarly, Kimoto et al. found that vitamin C in combination with a copper tripeptide (glycylglycylhistidine) was cytotoxic to Ehrlich ascites tumor cells in mice.69 In a short monograph, Morishige presented a case report of a patient with osteosarcoma successfully treated with ascorbate and the copper tripeptide, which was designed to mimic the copper transport site of albumin.70 The anticancer mechanism was hypothesized to be the release of copper locally by the high peptide-cleaving activity of tumor cells, thus generating cytotoxic radicals in the presence of ascorbate. A clever model using xenografts of human tumor fragments implanted in the subrenal capsule in mice has been used to demonstrate the immuneindependent inhibitory effect of vitamin C alone or combined with transition metal ions.71,72 Vitamin C was provided in the drinking water with copper or iron in order to generate oxidation products of vitamin C, which had more pronounced inhibitory effects than vitamin C itself. Prasad et al published two reviews that argued for the essentiality of multiple antioxidants, including large doses of vitamin C, in enhancing the cytotoxicity of chemotherapeutic drugs and radiation.73,74 In their in vitro experiments, the addition of vitamin C to the culture media increased the cytotoxicity of 5-fluorouracil, bleomycin, and sodium butyrate, as well as x-irradiation, in murine neuroblastoma cells compared to these agents without vitamin C. Vitamin C itself at low (5 µg/ml) and high concentrations (200 µg/ml) had no inhibitory effect on cell growth, although an inhibitory effect was observed in human melanoma cells at a high concentration of

vitamin C (100 µg/ml), but not at a low concentration (50 µg/ml). Whereas the addition of single antioxidants to the culture medium had variable effects on tumor cell growth, the most impressive results were produced by the combination of vitamins C and E, beta-carotene, and 13-cis retinoic acid. Of course, cell culture or animal experiments do not faithfully predict the outcome of the clinical application of vitamin C. However, abundant clinical evidence has been presented by Cameron and Pauling,75 Hoffer and Pauling,76 and Riordan77 detailing the therapeutic response of cancer patients to vitamin C given intravenously and/or orally or as part of a micronutrient regimen. Additionally, one small non-randomized clinical trial in Finland found that the provision of a micronutrient regimen, including large doses of vitamin C, in combination with chemotherapy and radiation significantly prolonged survival in patients with smallcell lung cancer compared to similar patients treated only with chemotherapy and radiation.78 The in vitro and in vivo data amassed to date do not seem to support the concern about the possible interference of vitamin C with standard therapy, as discussed above. However, definitive clinical data are lacking. In a commentary published in 2000, Padayatty and Levine called for a re-evaluation of vitamin C and cancer, especially when the vitamin is given intravenously, which results in plasma concentrations about ten times higher than orally administered vitamin C and comparable to media concentrations of vitamin C that demonstrated anticancer activity in vitro.79 While the failed Mayo Clinic trials of vitamin C and cancer80,81 have been criticized for other serious methodological flaws, Padayatty and Levine speculated that the critical difference between the work of Cameron and Pauling and the Mayo Clinic studies purportedly designed to replicate Cameron’s clinical work was the mode of administration, i.e. Cameron gave 10 grams/day or more of sodium ascorbate intravenously for about ten days followed by 180

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oral administration continued indefinitely, whereas the Mayo Clinic gave only oral vitamin C for a limited period. Recently, Tamayo and Richardson have reiterated the need for careful evaluation of vitamin C and cancer by carefully designed clinical trials that enroll patients with advanced common types of cancer and uncommon cancers and administer vitamin C intravenously or by bolus.82 Prevention or Treatment It is important to distinguish primary prevention studies from secondary prevention studies, which may be better termed “treatment” studies. The media do not often make this distinction when reporting clinical trials. Once a chronic disease has been established and reaches the point of symptomatic or clinical manifestation, it is useless to ask if the intervention with large doses of vitamin C will prevent disease. Such intervention will address only therapeutic response and the prevention of subsequent clinical events, and the result may be a consequence of the stage of the disease and the dose, duration, and mode of administration of vitamin C, among other factors. Indeed, if one wants to answer definitively the question, “Does vitamin C prevent heart disease?”, and if one plausibly assumes that vitamin C may be especially important in inhibiting the early stages of lesion development, a placebocontrolled, double-blind, randomized clinical trial with thousands of pre-adolescents (when lesions first appear) supplemented daily with various doses of vitamin C or placebo for three or four decades until clinical manifestations become apparent would need to be conducted. It is doubtful that such a study would ever be done. Likewise, one could determine if vitamin C prevents cancer in humans by designing a similar study and enrolling subjects before any cells have been initiated. Of course, it’s impossible to determine if one has any initiated cells and their precise fate. Several large, well-con-

trolled animal studies have demonstrated that vitamin C offers substantial protection against the development of mammary cancer in mice83 and skin cancer in mice,84,85 although it’s difficult to extrapolate to humans. Pharmacokinetics Very few careful pharmacokinetic studies on vitamin C in healthy humans have been published. Two German studies from 1970 helped to inform Linus Pauling’s opinion on the optimal intake of vitamin C.86,87 The first study of 75 healthy adults (25 women, 50 men) used oral doses of 1.5, 3, 6, and 12 grams of vitamin C and showed absorption of 49.5%, 36.2%, 25.6% and 16.1%, respectively. The mean excretion was 62% of the absorbed dose. The maximal plasma concentration of vitamin C at the largest dose was more than 114 µM. The companion study investigated the pharmacokinetics of intravenously administered vitamin C in 11 healthy adult men and 1 woman. Levine et al. published two pharmacokinetic studies, one with 7 healthy young men and the other with 15 healthy young women.29,30 The 1996 study with men demonstrated considerable interindividual variability. The vitamin C doses (30-2,500 mg/day) were administered sequentially over the study period; 6 men remained enrolled for the 1,000 mg/day dose and only 3 were available for determinations at the 2,500 mg/day dose. Nevertheless, Levine showed that lymphocytes became saturated with vitamin C at a dose of 100 mg/ day, which resulted in intracellular concentrations of about 3.5 mM. Neutrophil saturation also occurred at the 100 mg/day dose, which resulted in intracellular concentrations of 1.3 mM. Plasma levels of vitamin C increased rapidly between the 30 mg/day and 200 mg/day doses. Plasma concentrations of vitamin C were about 66 µM (range: 57.1-75.1 µM) at the 200 mg/day dose, 70 µM (range: 60.0-80.4 µM) at the 400 mg/day dose, and 77 µM (range: 70.5-84.3 µM) at the 1,000 mg/ day dose. There was a non-statistically

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significant increase at the 2,500 mg/day dose, although one of the three men showed an increase from 75.9 µM at the 1,000 mg/ day dose to 91.8 µM at the 2,500 mg/day dose. Levine’s recent study with women showed lymphocyte saturation at a daily dose of 200-400 mg, somewhat higher than the dose that saturated lymphocytes in men. The curve for plateau plasma vitamin C concentration as a function of dose in women was quite similar to that observed in men. Interestingly, over three weeks were required at a specific daily dose of vitamin C to achieve steady state plasma concentrations in men and over five weeks in women. It is not apparent that the investigators in the earlier German studies recognized this phenomenon. While these pharmacokinetic data are illuminating, they are not necessarily predictive of the pharmacokinetics in older or ill people whose need for ascorbate may be greater. A recent meta-analysis of 36 studies on the relationship between vitamin C intake and plasma concentrations found that the elderly (aged 60-96 years) exhibit a significantly lower plasma concentration of vitamin C than do adults (aged 15-65 years) for a given daily intake.88 For example, to achieve a vitamin C plasma concentration of 50 µM/L in 50% of adults, an estimated daily intake of 78 mg is needed, but the elderly need 150 mg. To achieve 50 µgM/L in 80% of adults requires an estimated daily intake of 173 mg, whearas the elderly need 478 mg. Indeed, it is difficult to reconcile some studies reporting benefits of large doses of vitamin C with Levine’s pharmacokinetic model. For example, Dawson et al. showed that supplementation with 1,000 mg/day of vitamin C, but not with 200 mg/day, dramatically reduced blood levels of lead in adult male smokers by 81% after one week.89 An observational study by Cheng et al. found that older men whose intake of vitamin C was at least 339 mg/day had lower lead levels in their blood than men whose intake was less than 109 mg/day.90 Johnston

reported that an intake of 2,000 mg/day of vitamin C, resulting in a plasma concentration of 1.02 mg/100 ml, substantially reduced blood histamine levels from 72.3 ng/ml at baseline to 43.1 ng/ml, whereas a 500 mg/day dose of vitamin C, resulting in a plasma concentration of 0.95 mg/100 ml, had little effect on histamine levels.91 This observation may help explain why large doses of vitamin C have a salubrious effect on common cold symptoms, whereas small doses have little discernable effect. These reports also highlight the importance of dose and duration in evaluating the preventive role of vitamin C and, additionally, the mode of administration in assessing therapeutic response. Special Experimental Considerations A number of interesting observations about vitamin C used in in vitro and in vivo models are important to consider when designing or evaluating experimental studies. Many cultured cells without vitamin C added to the medium are scorbutic and do not reflect physiological status. For example, human endothelial cells cultured without vitamin C added to the medium show abnormally high levels of oxidative stress that affect other parameters, such as eNOS activity. 92 Small amounts of supplemental vitamin C (0.076% by weight) administered to mice, a species able to synthesize vitamin C, depress levels of vitamin C in the liver, lung, muscle, spleen, and plasma compared to unsupplemented mice, demonstrating the so-called “mouse effect.”93 To raise the vitamin C level in these tissues (except plasma) in mice, the amount of vitamin C added to the diet must be greater than 0.5% by weight. Additionally, guinea pigs, an excellent animal model for studying vitamin C because of their inability to synthesize the vitamin, maintained on vitamin C deficient diets and housed in cages with wood chips can remain ascorbutic by consuming the chips, which 182

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provide enough vitamin C to prevent scurvy.94 The Real Problem The magnitude of the precise prophylactic effect of vitamin C against heart disease and cancer remains unresolved, and we have little dose response data on the effect of vitamin C on its manifold biochemical reactions. Studies using the guinea pig model have demonstrated remarkable protective effects of high-dose vitamin C against liver damage and mortality caused by aflatoxin B1 exposure95 and against neuro-lathyrism or death induced by exposure to ß-N-oxalylamino-Lalanine, a potent neurotoxin.96 These results, combined with the aforementioned human studies on lead and vitamin C, show the potential for substantial improvements in public health in regions with high toxicant exposure. We know that vitamin C has an important and underappreciated role in clinical practice and that it is a very safe substance. Studies will continue to report the gamut of beneficial, neutral, or potentially harmful effects of supplemental vitamin C, which must be reviewed carefully and critically. Many, perhaps most, of these studies will be imperfect, containing insignificant flaws or serious problems that render the results questionable. In particular, the news media must endeavor to display much more scientific competence, perspective and technical precision in their coverage of scientific and medical reports on vitamin C. Otherwise, the public may become confused and needlessly suffer. As Wakimoto and Block pointed out, about 50% of older Americans ingest less than the RDA for vitamin C (90 mg/day for men, 75 mg/ day for women), and about 25% ingest less than 50% of the RDA for vitamin C.97 Clearly, inadequate daily intake of vitamin C is among the most serious problems that need to be addressed.

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smokers and their role in ascorbate free radical formation in plasma. Free Radic Res, 2001; 34(3): 209-219. 32. Proteggente AR, Rehman A, Halliwell B, et al: Potential problems of ascorbate and iron supplementation: pro-oxidant effect in vivo? Biochem Biophys Res Commun, 2000; 277(3): 535-540. 33. Lee SH, Oe T, Blair IA: Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins. Science, 2001; 292: 20832086. 34. Sattler W, Maiorino M, Stocker R: Reduction of HDL- and LDL-associated cholesterylester and phospholipid hydroperoxides by phospholipid hydroperoxide glutathione peroxidase and Ebselen (PZ 51). Arch Biochem Biophys, 1994; 309(2): 214-221. 35. Zamburlini A, Maiorino M, Barbera P, et al: Measurement of lipid hydroperoxides in plasma lipoproteins by a new highly-sensitive ‘single photon counting’ luminometer. Biochim Biophys Acta, 1995; 1256: 233-240. 36. Suh J, Zhu B-Z, Frei B: Ascorbate does not act as a pro-oxidant towards lipids and proteins in human plasma exposed to redox-active transition metal ions and hydrogen peroxide. Free Radic Biol Med, 2003; 34(10): 1306-1314. 37. Chen K, Suh J, Carr AC, et al: Vitamin C suppresses oxidative lipid damage in vivo, even in the presence of iron-overload. Am J Physiol Endocrinol Metab, 2000; 279: E1406-E1412. 38. Dwyer JH, Nicholson LM, Shirecore A, et al: Vitamin C supplement intake and progression of carotid atherosclerosis, the Los Angeles Atherosclerosis Study. 40th Annual Conference on Cardiovascular Disease Epidemiology and Prevention, La Jolla, CA, 2000. 39. Kritchevsky SB, Shimakawa T, Tell GS, et al: Dietary antioxidants and carotid artery wall thickness. Circulation, 1995; 92: 2142-2150. 40. Fang JC, Kinlay S, Beltrame J, et al: Effect of vitamins C and E on progression of transplantassociated arteriosclerosis: a randomized trial. Lancet, 2002; 359: 1108-1113. 41. Salonen RM, Nyyssonen K, Kaikkonen J, et al: Six-year effect of combined vitamin C and E supplementation on atherosclerotic progression. Circulation, 2003; 107: 947-953. 42. Salonen JT, Nyyssonen K, Salonen R, et al: Antioxidant supplementation in atherosclerosis prevention (ASAP) study: a randomized trial of the effect of vitamins E and C on 3-year progression of carotid atherosclerosis. J Intern Med, 2000; 248: 377-386. 43. Brown BG, Zhao, X-Q, Chait, A, et al: Simvistatin and niacin, antioxidant vitamins, or the combination for the prevention of coro-

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